Process for the observation of at least one sample region with a light raster microscope with light distribution in the form of a point

ABSTRACT

Process for the observation of at least one sample region with a light raster microscope by a relative movement between the illumination light and sample via first scanning means along at least one scanning axis essentially perpendicular to the illumination axis wherein the illumination light illuminates the sample in parallel at several points or regions and several points or regions are detected simultaneously wherein at an angle to the plane of the relative movement, preferably perpendicular thereto, second scanning means are moved and an image acquisition takes place by the movement of the first and second scanning means being coupled and a three-dimensional sampling movement being done by the illumination of the sample wherein the second scanning means are coupled to the movement of the first scanning means in such a manner that straight and/or curved lines and/or plane and/or curved surfaces are scanned which are extended along at least one scanning direction of the first scanning means as well as along the scanning direction of the second scanning means.

STATE OF THE ART

In DE 19702753A, DE 19702752A, U.S. Pat. No. 6,037,583 laser scanningmicroscopes are described.

Along with the X/Y movement of the light point over the sample, whichusually is produced by galvanoscanners, it is a known practice to alsoperform a z-movement in order to acquire image stacks in depth.

This movement in the z-direction is done, for example, mechanically bymovement of the objects by means of a z-movement of the sample tray(piezo tray, HRZ tray in DE 19829982A1) or also by movement of theobjective (for example, objective with piezo drive).

Here, in the first case, a target position in the z-direction can bepredefined, where a measured value acquisition occurs after ending thefeed movement of the z-drive or, in the second case, there is acontinuous movement in the z-direction, where after the termination of arun-in phase of the positioning movement of the z-drive (accelerationphase) a measured value acquisition is done continuously ordiscontinuously.

The first variant is very slow.

Variant 2 does, in fact, make possible, for example, the rapidacquisition of a z-stack. Between sequential z-scans, however, a pausefor braking or accelerating of the z-drive is needed until thepredefined movement has begun.

INVENTION

Through the invention, as described in the following, in particular inthe independent and subordinate claims, a rapid z-focus is produced bythe operation of the z-drive via, in particular, periodic controlsignals, with coordination of the measured value acquisition and thecontrol of the z-drive based on the mechanical and electrical propertiesof the system.

The z-movement is done advantageously by movement of the objective viapiezo drive or galvanometer drive or by z-movement of the sample viapiezo drive or galvanometer drive.

For the particularly rapid z-movement of the light spot, adaptive opticsin the illumination beam path of the microscope are also suitable. Thesemove the light spot in the sample by defocusing in the z-direction(DE19733193A1).

According to the invention different control modes for the z-movementcan be realized.

The scanner control for x and y is, for example, described in DE19702752A.

In FIG. 5 a linear control (control signal A) with rapid sample movementP (high frequency) is represented as a function of time.

The movement P is distorted by electrical and mechanical properties ofthe system (for example, inertia, resonance frequencies).

Here it is represented that despite linear control as of a frequency oftypically 1 Hz or more the resulting z-movement runs with undefinedcurve form.

In FIG. 6 an advantageous linearization of the z-scanning movement isthus represented.

A pre distorted control A adapted to the system is represented. Themovement P is linear after transmission through the system (up to smallreversing areas).

That is achieved by the periodic control signal being distorted in sucha manner that the distortions characteristic for the system in question,which are related to the non-ideal electrical and mechanicaltransmission of the control signals, are compensated.

The control curves can, for example, be determined empirically inadvance with the aid of values from position sensors which register thescanner position and by the inclusion of error curves and selectivecorrection.

Likewise, the transmission function of the system can be measureddirectly with the aid of values from position sensors which register thescanner position. The transmission function of the system gives for eachfrequency used the deviation of the real movement between the sample andthe light spot from the predefinitions by the control signals. For eachfrequency there is a phase shift (for example, delay due to thetransmission) and an amplitude change (for example, damping due to thetransmission).

The knowledge of the transmission function of the system makes possiblethe selective calculation of a desired curve for the movement betweenthe sample and the light spot in a suitable control curve for thegeneration of this movement. The empirical determination of the suitablecontrol curve or also the selective calculation of the control curve canadvantageously be done by a suitable Fourier series expansion of thecontrol signal and the variation of the Fourier coefficients. Dependingon the precision demanded in the case in question, the coefficientsabove the limiting frequency can be left out of consideration.

The position sensors which register the scanner position can, forexample, be optical or capacitive.

In order to achieve, in z, a movement curve P for the sample whichexactly corresponds to the predefinition (for example, exactly linearmovement, that is, constant speed in the range used), a pre-distortionof the periodic control signal of the z-drive (for example, piezo) isdone in such a manner that the mechanical movement corresponds exactlyto the desired course of movement (for example, linear curve over time).

The distortions occurring depending on the frequency composition of thecontrol signal and due to the frequency-dependent transmission functionsof the z-drive (differences between control signal curve (predefinition)and movement curve) are compensated in so doing.

The measured value acquisition by the laser scanning microscope is thendone, for example, at temporally constant intervals (reversing areas ofthe scanning movement excepted).

The linearization (determination of the pre-distortion) is done, forexample, via Fourier coefficients.

The generation of the scanner control signals is done via a Fourierexpansion:

[see original for equation]

-   -   a₁, a₃, a₅, . . . : amplitude coefficients    -   φ₁, φ₃, φ₅, . . . : phase coefficients    -   f: ground frequency of the scanning movement    -   t: time    -   Pos(t): deflection of the scanner mirror

The individual coefficients for the amplitudes and phases (a₁, φ₁) servefor the calibration of the movement of the scanner.

In the case of an (in practice not possible) ideal, distortion-freeconversion of the control signals into the scanning movement, the idealFourier series of the delta function a₁=1, φ₁=0(i=1,3,5,7, . . . )

would result for the movement in the form of a symmetric delta curve.

The individual frequency components of the control signal are typicallytransmitted by the scanning system nearly independently of one another(depending on the linearity of the system).

In practice an ideal delta curve for the deflection of the scannermirror cannot be implemented.

This would have infinitely high accelerations of the scanner mirror atthe reversing points as a consequence.

Thus, we restrict ourselves in the scanning of an image (line-by-linescanning of a sample region) to a usable range of the movement of thescanner. The braking and acceleration of the mirror is done outside ofthis range.

Typical for scanning microscopy are usable ranges of ca. 85% of thescanning period.

Advantageously a simple and selectively directed calibration ispossible.

Alternatively, a linearization of the movement of the scanner can alsobe done via a control curve which is variable for calibration andgenerated directly through look-up tables. Advantageously, this variablecontrol curve can also be generated through variable splines.

These are predefined measurement points whose rise in their vicinity ispredefined in addition, which leads to a smoother form of the curve.

A calibration of the control curves can also be done via so-called FITTalgorithms (variation of the look-up tables up to which movement islinear).

The calibration can be done via position or speed sensors installed inthe movement system (for example, electrical or optical position sensorsinstalled in the z-drive) or via an optical evaluation of a signal (3Dcalibration sample, for example, equidistant scattering or reflectingplanes in the sample or a sample grid) generated (for example,re-scattered) by a calibration sample.

A calibration can also be done via an external measuring system (forexample, electronic μm clock on the sample tray or on the objective).

In FIG. 7 an additional control mode is represented.

A periodic z-movement occurs whose form can be adapted to the mechanicalor electrical limits of the system “moved mass+drive”.

The advantage is a lower mechanical load on the sample and system.

This can, for example, advantageously be a sinusoidal scanning movement.

The operation of the system “moved mass+drive” can in this case also bedone in resonance, where previously the resonant frequency is determinedempirically. Thereby very high amplitudes at high frequencies are madepossible.

A measured value acquisition by the laser scanning microscope can, forexample, be done at temporally or positionally constant intervals,where, for example, the measured value is triggered with an installedposition sensor.

That means that there is always measurement when the desired z-positionis reached (or, for example, an x-y scan is triggered). For a temporallycontrolled measured value acquisition, a calibration carried out inadvance supplies the association of measurement time points andz-position (for example, by means of a look-up table).

A defined, calibrated control of the individual scanning axes in thesystem makes possible in an advantageous manner a synchronous control ofthe desired scanning axes and the a selective control of the directionof movement of the light spot in the sample volume.

The points in time for the measured value acquisition could also becalculated from the control curve.

A measured value acquisition can be done at temporally constantintervals.

A retroactive de-distortion of the image or z-image stack on a linearmovement curve is done by means of a calibration carried out in advanceor with the aid of position measurements taken synchronously to themeasured values.

Generally the measured value acquisition can also be donebi-directionally (forward and backward direction) to accelerate theimage acquisition in periodic scanning movements (in x, y, and z).

Advantageous scanning strategies for the advantageous combination of thethree fast axes x, y, z are explained in more detail in the followingwith the aid of the schematic representation in FIG. 4.

In FIG. 8 an X/Y sampling is represented schematically.

A scanner (X) makes possible by control with a variable control voltagethe free positioning of a laser spot in the sample along a line(normally a straight line) (X_(min) . . . , X_(max)).

With a second scanner (Y) (axis of rotation in an advantageous mannerperpendicular to the axis of rotation of the first scanner) the spot canbe positioned freely within a surface (normally a plane=scanning plane)(X_(min)<X<X_(max), Y_(min)<Y<Y_(max)).

In order to traverse to a predefined position within the scanning plane,the first scanner (X) is controlled with the position voltage whichcorresponds to the projection of the target point on its positioningdevice (X_(target)). Simultaneously, the second scanner is alsocontrolled with the position voltage which corresponds to the projectionof the target point on its positioning device (Y_(target)).

In FIG. 9 it is represented how by means of the coordinated synchronouscontrol of the X-scanner and Y-scanner a scanning field SC oblique tothe x-axis an y-axis can be generated by each of the X-scanner andY-scanner being moved simultaneously and with uniform speed whentraversing a scanning line.

The angle of the obliqueness is set by setting the amplitude ratio ofthe voltage signal of the scanners.

With a third scanner (Z) (axis of rotation advantageously perpendicularto the axis of rotation of the first scanner and perpendicular to theaxis of rotation of the second scanner) the spot can be positionedfreely within a sample volume (scanning volume) limited by the maximalangle of deflection of the scanners (X_(min)<X<X_(max),Y_(min)<Y<Y_(max), Z_(min)<Z<Z_(max)).

In order to traverse to a predefined position within the scanningvolume, all three scanners (X, Y, and Z) are simultaneously (analogousto the case with 2 scanners) controlled with the position voltage whichcorresponds to the projection of the target point on the positioningdevice of the respective scanner (X_(target), Y_(target), Z_(target)).

By traversing a sequence of target positions, sample regions which canbe defined in advance can be traversed.

The sample regions can be one-dimensional, two-dimensional, orthree-dimensional. Sequential traversing of points which lie on a line(for example, straight lines) corresponds to a line scan (scanning of aline) (one-dimensional).

If only one scanner is moved, e.g. the X-scanner, a straight line isacquired which is parallel to the direction of movement of the scanner.

A line scan rotated in the plane is generated by synchronized movementof two scanners (see FIG. 5). Arbitrarily rotated straight lines, e.g.in the XY plane, can be generated.

By sequential scanning with simultaneous movement of two scanners aclosed, arbitrarily formed line (spline) can be generated.

Sequential traversing of several lines, one after the other, generates asurface scan.

Often the lines lie within a plane—a two-dimensional plane scan is done.

A sequential traversing of straight lines in the direction of movementof the scanners (rectangular scan) is customary. A scanner is in thiscase moved rapidly (X, line scanner) and after each line the otherscanner (Y, image scanner) is moved a bit further (change of the offset)and thus an offset between the lines is generated perpendicular to thelines.

An arbitrarily rotated rectangle in a plane can be generated (FIG. 9).

A sequential traversing of several plane scans, where, for example, onelies over the other, generates a volume scan (three-dimensional).

Advantageous processes and modes of action in the case of the lightspot's movement which is in three spatial coordinates, coordinated withone another, and rapid according to the invention is intended to beexplained in the following.

FIG. 10 shows schematically the position of a light spot in the X/Y/Zspace.

The position of the light spot is advantageously changed by control ofthe scanners which are coordinated with one another as follows:X-scanner, Y-scanner, and Z-scanner scan synchronously (see figure!).

At the point in time t=T1 the three scanners are at the start point ofthe line (X1, Y1, Z1)

-   X-scanner is at X=X1-   Y-scanner is at Y=Y1-   Z-scanner is at Z=Z1

In order to achieve a straight-line movement of the spot in the samplevolume, the form of the control signal of all three scanner is chosen sothat in an individual control of the scanners a saw-tooth or triangularform movement of the spot in the sample would arise (possibly thecontrol signals must be pre-distorted).

The selective deflection is done, therefore, relative to the zero axisof the vibration, either symmetrically upwards and downwards(bidirectional scan) or in the reverse direction of the movement steeper(saw-tooth) for a unidirectional scan in which traversing back into thestarting positions should be as rapid as possible.

At the start point (X1, Y1, Z1) all three scanners (at the same time)are at an extreme point (reversing point) of their periodic movement.

All three scanners run with the same ground frequency, that is, afterthe time Tp (period time) all three scanners are once again at theirstarting point (starting point of the line).

The amplitude of the scanning movement is chosen so that after theexpiration of the half period (that is, when all three scanners havearrived at their periodic movement's extreme point opposite theirstarting point, time point t=T2), all three scanners appear at the endpoint of the line (X2, Y2, Z2).

-   X-scanner is at X═X2-   Y-scanner is at Y═Y2-   Z-scanner is at Z=Z2

That means that the amplitude X-scanner ═X2-X1, amplitude Y-scanner═Y2-Y 1, and amplitude Z-scanner=Z2-Z1.

This form of control causes the scanning of a straight line from point(X1, Y1, Z1) to point (X2, Y2, Z2).

The starting point and ending point can be freely chosen within thesample volume, that is, the length and the orientation of the line canbe freely chosen.

For arbitrary alignment of the scanning direction in the sample volume,there is a synchronous control of all three scanners analogous to thesynchronous control of the two scanning axes for rotation of thedirection of scanning in the plane.

In the case of an HRZ tray a movement of the sample tray is done with agalvanoscanner which advantageously according to the invention iscontrolled as the x/y scanner.

The current through the operating coil of the galvanoscanner iscontrolled by means of regulation electronics with the aid of theposition sensor in such a manner that the totality of the regulationcircuit and scanner traverses to the position which is predefined bymean of a control signal.

In the case of a resonance scanner the input quantity for the regulationcircuit is then not the position+theoretical value (control signal) butrather typically the amplitude (sensor) and theoretical value.

One can naturally also move the objective with a galvanoscanner or witha resonance scanner (the weight of the objective then determines theresonance frequency!).

In the case of a movement of the sample tray of the objective with apiezo element a change of the position is accomplished by changing thehigh voltage applied to the piezo.

Since the regulation circuits can be too slow or produce errors, theremust be calibration in both cases, at least as of certain speeds, thatis, the control signals (=theoretical values for the electronicregulation circuits) are changed (pre-distorted) so that the actualmovement corresponds to the desired movement.

A synchronous, rapid control in three scanning axes (x, y, and z)advantageously makes possible an arbitrary orientation of the directionof scanning in the sample volume. In this way the direction of scanningcan be adapted to the characteristics of the samples.

By way of example, a line scan oriented arbitrarily in the sample spaceis generated, which advantageously can be aligned to a prominent samplestructure.

Furthermore, a surface scan oriented arbitrarily in the sample space canbe generated which advantageously can be aligned to a sample surface.

A sequential scanning of lines is done in a plane lying arbitrarily inthe sample space, where an offset between the lines can advantageouslybe perpendicular to the lines and equidistant.

An example would be the scanning of a plane which extends over a rangein z.

The scanning surface can be arbitrarily tilted and turned by rapidcontrol of three scanners.

The direction of scanning and form of scanning can be adapted to theposition and the form of the sample in all three axes, for example, openor closed spline scans with arbitrary curve in the sample space.

The scanning surface can also be arched with appropriate control of thescanners.

Line scans can likewise be correspondingly arched.

In all three axes bidirectional scanning is also possible by theperiodic control for the increase of the imaging rate.

Instead of the bidirectional scanning, closed lines formed arbitrarilyin space can be scanned (corresponds to a periodic movement of all threescanners). Through sequential scanning of such closed lines, arbitrarilyformed surfaces in the sample volume can be scanned.

In the case of a line scanner, a coordinate can be chosen less freely.

In the case of line scanners the line is predefined as a scan form andis not generated by a scanning axis.

In the following a scanning microscope, in particular a line scanner, isexplained in more detail with the aid of additional representations.

FIG. 1 shows schematically a laser scanning microscope 1 which isassembled essentially from five components: a radiation source module 2which generates excitation radiation for the laser scanning microscope,a scanning module 3 which conditions the excitation radiation andsuitably deflects it for scanning over a sample, a microscope module 4showed only schematically for simplification, said microscope moduledirecting the scanning radiation provided by the scanning module in amicroscopic beam path onto a sample, and a detector module 5 whichreceives and detects the optical radiation from the sample. Along withthis, the detector module 5 can, as is represented in FIG. 1, beimplemented with multiple spectral channels.

For the general description of a laser scanning microscope which scanspoint-by-point, reference is made to DE 19702753A1 which is thus acomponent of the present description.

The radiation source module 2 generates illumination radiation which issuitable for laser scanning microscopy, that is, in particular,radiation which can resolve fluorescence. Depending on the application,the radiation source module comprises for this purpose several radiationsources.

In a form of embodiment represented, two lasers 6 and 7 are provided inthe radiation source module 2 after each of which a light valve 8 aswell as an attenuator 9 is connected and which couple their radiationvia a coupling point 10 into a light guide fiber 11. The light valve 8acts as a beam deflector with which shutting off of the beam can beeffected without the operation of the laser itself in the laser unit 6or 7 having to be switched off.

The light valve 8 is, for example, formed as AOTF which, for shuttingoff a beam, deflects the laser beam before its coupling into the lightguide fiber 11 in the direction of a light trap not represented.

In the exemplary representation of FIG. 1 the laser unit 6 comprisesthree lasers B, C, and D, in contradistinction to which the laser unit 7contains on one laser A. The representation is therefore exemplary of acombination of individual and multi-wave length lasers which are coupledindividually or also jointly to one or more fibers. Also, the couplingcan be done simultaneously via several fibers whose radiation, afterrunning through adaptation optics, is later mixed by color combiners. Itis thus possible to use the most varied wave lengths or ranges for theexcitation radiation.

The radiation coupled into the light guide fiber 11 is combined by meansof displaceable collimation optics 12 and 13 via beam-combining mirrors14, 15 and changed with regard to its beam profile in a beam-formingunit.

The collimators 12, 13 provide for the radiation supplied by theradiation source module 2 to the scanning module 3 being collimated intoan infinite beam path. This is done in each case advantageously with asingle lens which has a focusing function by displacement along theoptical axis under the control of a (not represented) central controlunit by the distance between the collimators 12, 13 and the respectiveend of the light guide fiber being variable.

The beam-forming unit, which will be explained in still more detaillater, generates from the rotationally symmetric beam profiled in aGaussian shape, as is present after the beam—combining mirrors 14, 15, alinear beam which is no longer rotationally symmetric but rather issuitable in cross-section for the generation of a rectangularlyilluminated field.

This illumination beam, also designated as linear, serves as excitationradiation and is conducted via a principal color splitter 17, and stillto be described zoom optics, to a scanner 18. The principal colorsplitter will be discussed in more detail later. Here let it merely bementioned that it has the function of separating sample radiationreturning from the microscope module 4 from the excitation radiation.

The scanner 18 deflects the linear beam monoaxially or diaxially,according to which it is bundled by a scanning objective 19 as well as atubular lens and an objective of the microscope module 4 into a focus 22which lies in a preparation or in a sample. In so doing, the opticalimaging is done so that the sample is illuminated with excitationradiation in a focal line.

Fluorescence radiation excited in the linear focus in such a mannerreaches, via the objective and tubular lens of the microscope module 4and the scanning objective 19, back to the scanner 18 so that in thereverse direction, toward the scanner 18, an inactive beam is presentonce more.

One thus also speaks of the fact that the scanner 18 descans thefluorescence radiation.

The principal color splitter 17 permits the fluorescence radiation inwave length ranges other than the excitation radiation to pass so thatit is reversed via a reversing mirror 24 in the detector module 5 andthen can then be analyzed. The detector module 5 comprises in the formof embodiment of FIG. 1 several spectral channels, that is, thefluorescence radiation coming from the reversing mirror 24 is split in asecondary color splitter 25 into two spectral channels.

Each spectral channel has a slotted diaphragm 26 which realizes aconfocal or partially confocal imaging with respect to the sample 23 andwhose magnitude determines the depth resolution with which thefluorescence radiation can be detected. The geometry of the slotteddiaphragm 26 thus determines the sectional plane within the (thick)preparation from which fluorescence radiation is detected.

Behind the slotted diaphragm 26, a block filter 27 is also disposedwhich blocks undesired excitation radiation arriving at the detectormodule 5. The radiation which is separated off in that manner, comesfrom a certain depth section, and is expanded so as to be linear, isthen analyzed by a suitable detector 28. Set up analogously to the colorchannel described is the second spectral detection channel which alsoincludes a slotted diaphragm 26 a, a block filter 27 a, and a detector28 a.

The use of a confocal slot aperture in the detector module 5 is onlyexemplary. Naturally a single-point scanner can also be realized. Theslotted diaphragms 26, 26 a are then replaced by aperture shields andthe beam-forming unit can be omitted. Otherwise, for such a mode ofconstruction, all the optical elements are implemented to berotationally symmetric. Then, naturally, instead of a single-pointsampling and detection, arbitrary multi-point arrangements, such aspoint clouds and Nipkow disk concepts, can, in principle, be used, aswill be explained later with the aid of FIGS. 3 and 4. However, it isthen essential that the detector 28 is spatially resolving since thereis a parallel observation of several sample points during the pass ofthe scanner.

In FIG. 1 it is to be seen that the Gaussian beam bundles present behindthe movable, i.e. displaceable, collimators 12 and 13 are combined via amirror arrangement in the form of the beam combining mirrors 14, 16 and,in the mode of construction shown, are subsequently converted, with aconfocal slotted diaphragm, into a beam bundle with rectangular beamcross-section.

In the form of embodiment of FIG. 1 a cylinder telescope 37 is used inthe beam-forming unit behind which an aspherical unit 38 is disposedwhich is followed by cylinder optics 39.

After the reforming there is a beam which illuminates, in a profileplane, essentially a rectangular field, where the intensity distributionalong the field axis is not Gaussian in form but rather chest-shaped.

The illumination arrangement with the aspherical unit 38 can serve forthe uniform filling of a pupil between a tubular lens and an objective.With this, the optical resolution of the objective can be fullyexploited. This variant is thus also expedient in a single-point ormulti-point scanning microscope system, e.g. in a line-scanning system(in the latter in addition to the axis in which focusing is in or on thesample).

The excitation radiation conditioned, e.g. to be linear, is deflectedonto the principal color splitter 17. This is implemented, in apreferred form of embodiment, as a spectral-neutral splitter mirroraccording to DE 10257237 A1 whose disclosure is included here in itsfull extent. The term “color splitter” therefore also includes splittersystems acting in a non-spectral manner. Instead of the describedspectrally independent color splitter a homogeneous neutral splitter(for example, 50/50, 70/30, 80/20, or the like) or a dichroitic splittercan be used. So that an application-dependent choice is possible, theprincipal splitter is preferably provided with mechanics which makepossible simple exchange, for example, by a corresponding splitter wheelwhich contains individual, exchangeable splitters.

A dichroitic principal splitter is particularly advantageous whencoherent, i.e. directed, radiation is to be detected, such as, forexample, reflection, Stokes or anti-Stokes Raman spectroscopy, coherentRaman processes of higher order, generally parametric non-linear opticalprocesses such as second harmonic generation, third harmonic generation,sum frequency generation, and two and multi-photon absorption orfluorescence. Several of these processes from non-linear opticalspectroscopy require the use of two or more laser beams which aresuperimposed collinearly. In this case the represented beam combinationof the radiation of several lasers has proven itself particularlyadvantageous. Basically the dichroitic beam splitter widely used influorescence microscopy can be used. Also, for Raman microscopy it isadvantageous to use holographic notch splitters or filters in front ofthe detectors to suppress the Rayleigh scattering. In the form ofembodiment of FIG. 1 the excitation radiation or illumination radiationis supplied to the scanner 18 via zoom optics 41 under motor control.With this, the zoom factor can be adapted and the sample viewing fieldcan be varied continuously in a certain range of displacement.Particularly advantageous is zoom optics in which, during adaptation ofthe focus position and the imaging scale, the pupil position remains ina continuous adjustment process. The zoom optics 41's three motordegrees of freedom represented in FIG. 1 and symbolized by an arrowcorrespond precisely to the number of degrees of freedom which areprovided for the adaptation of the three parameters, imaging scale,focus position, and pupil position.

Particularly preferred is zoom optics 41 at whose output-side pupil afixed aperture 42 is disposed. In a practical, simple realization, theaperture shield 42 can also be predefined by the limiting of the mirrorsurface of the scanner 18. The output-side aperture 42 with the zoomoptics 41 achieves the result that independently of the setting of thezoom enlargement a fixed pupil diameter is always imaged on the scanningobjective 19. Thus, the objective pupil also remains completelyilluminated at any setting of the zoom optics 41. The use of astand-alone aperture shield 42 advantageously prevents the occurrence ofunwanted scattering radiation in the area of the scanner 18.

The cylinder telescope 37 works together with the zoom optics 41, saidcylinder telescope also being actuable by motor and disposed in front ofthe aspherical unit 38. This is, in the form of embodiment of FIG. 2,chosen for reasons of a compact design but does not have to be so.

If a zoom factor smaller than 1.0 is desired, the cylinder telescope 37is automatically pivoted into the optical beam path. It prevents theaperture shield 42 from being completely illuminated when the zoomobjective 41 is reduced. The pivotable cylinder telescope 37 thusensures that even at zoom factors less than 1, i.e. independently of thesetting of the zoom optics 41 at the position of the objective pupil, anillumination line of constant length is always present. In comparison toa simple viewing field zoom, laser power losses in the illumination beamare avoided.

Since on pivoting the cylinder telescope 37 a jump in image brightnessin the illumination line is unavoidable, it is provided in the (notrepresented) control unit that the feed rate of the scanner 18 or anamplification factor of the detectors in the detector module 5 isadapted accordingly when the cylinder telescope 37 is activated in orderto hold the image brightness constant.

Along with the motor-driven zoom optics 41 as well as motor-activatablecylinder telescope 37, remote-controlled adjust elements are alsoprovided in the detector module 5 of the laser scanning microscope ofFIG. 1. For the compensation of color length errors, for example, roundoptics 44 as well as cylinder optics 39 are provided in front of theslotted diaphragm and cylinder optics 39 are provided directly in frontof the detector 28, said cylinder optics always being displaceable bymotor in the axial direction.

In addition, a correction unit 40 is provided for compensation, saidcorrection unit being described briefly in the following.

The slotted diaphragm 26 forms, together with round optics 44 disposedin front, as well as the first cylinder optics 39 also disposed infront, as well as the second cylinder optics 39 disposed behind, apinhole objective of the detector arrangement 5, where the pinhole hereis realized by the slotted diaphragm. In order to avoid an undesireddetection of excitation radiation reflected in the system, the blockfilter 27 is introduced in front of the second cylinder lens 39, saidblock filter having suitable spectral properties in order to allow onlydesired fluorescence radiation to reach the detector 28, 28 a.

A replacement of the color splitter 25 or the block filter 27 isassociated unavoidably with certain tilting or wedging errors onpivoting in. The color splitter can leave a fault between the sampleregion and the slotted diaphragm 26. The block filter 27 can leave afault between the slotted diaphragm 26 and the detector 28. In order toprevent a new adjustment of the position of the slotted diaphragm 26 orthe detector 28 from being necessary, a plane-parallel plate 40 isdisposed between the round optics 44 and the slotted diaphragm 26, thatis, in the imaging beam path between the sample and the detector 28,where said plate can be brought under the control of a controller invarious tilting settings. The plane-parallel plate 40 is, for thispurpose, mounted, in such a manner that it can be displaced, in asuitable holder.

FIG. 2 shows how a region of interest (ROI) can be chosen, with the aidof the zoom optics 41, within the maximum available viewing field SF. Ifone leaves the control of the scanner 18 so that the amplitude does notchange, as is necessarily required, for example, in resonance scanners,an enlargement set on the zoom optics of greater than 1.0 causes anarrowing of the chosen region ROI centered about the optical axis ofthe scanning field SF.

Resonance scanners are, for example, described in Pawley, Handbook ofBiological Confocal Microscopy, Plenum Press 1994, Page 461 ff. If onecontrols the scanner so that it samples a field asymmetrically relativeto the optical axis, that is, to the resting position of the scannermirror, then one obtains in connection with a zoom action an offsetdisplacement OF of the chosen region ROI. To descan due to the alreadymentioned action of the scanner 18, and due to the repeated pass throughthe zoom optics 41, the choice of the interesting region ROI in thedetection beam path is once again cancelled in the direction of thedetector. Thus, one can make an arbitrary choice, lying within thescanning image SF, for the region ROI. In addition, one can obtainimages for different choices of the region ROI and then combine them toform a highly resolved image.

If one would like to displace the chosen region ROI not only by anoffset OF with respect to the optical axis but rather also in additionto rotate it, a form of embodiment is expedient which provides, in apupil of the beam path between the principal color splitter 17 and thesample 23, an Abbe-König prism which, in a known manner, has, as aconsequence, a rotation of the image.

This is also cancelled in the direction of the detector. Then one canmeasure images with different offset displacements OF and differentangles of rotation and subsequently enhance it computationally to form ahighly resolved image, for example, according to an algorithm as isdescribed in the publication Gustafsson, M., “Doubling the lateralresolution of wide-field fluorescence microscopy using structuredillumination” in “Three-dimensional and multidimensional microscopy:Image acquisition processing VII”, Proceedings of SPIE, Vol. 3919(2000), p. 141-150.

In DE 10257237A1 additional ROI images with a line scanner are disclosed(change effective focal length, mechanical aperture shields).

FIG. 3 shows an additional possible mode of construction for a laserscanning microscope 1 in which a Nipkow disk approach is realized. Thelight source module 2, which is represented in FIG. 3 in very simplifiedform, illuminates, via a mini lens array 65 through the principal colorsplitter 17, a Nipkow disk 64, as, for example, is described in U.S.Pat. No. 6,028,306, WO 88 07695, or DE 2360197 A1. The Nipkow disk'spinholes illuminated via the mini lens array 65 are imaged in the samplelocated in the microscope module 4. In order to be able to vary thesample-side image size, zoom optics 41 are once again provided.

In the conversion to the mode of construction of FIG. 1 the illuminationis done, in the case of the Nipkow scanner, in the pass through theprincipal color splitter 17 and the radiation to be detected is mirroredout. Moreover, in the conversion to FIG. 2, the detector 28 isimplemented to be spatially resolving so that the multi-pointillumination achieved with the Nipkow disk 64 is also accordinglysampled in parallel. Furthermore, between the Nipkow disk and the zoomoptics 41, suitable fixed optics 63 with positive refractive power isdisposed which converts the radiation, exiting through the pinholes ofthe Nipkow disk 64 in a diverging manner, into a suitable bundlediameter. The principal color splitter 17 is, for the Nipkow layout ofFIG. 3, a classical dichroitic beam splitter, that is, not thepreviously mentioned beam splitter with reflecting area in the form of aslot or point.

Zoom optics 41 corresponds to the previously explained mode ofconstruction where naturally the scanner 18 is superfluous due to theNipkow disk 64. It can nonetheless be provided if one would like tocarry out the choice of a region ROI explained with the aid of FIG. 2.The same applies for the Abbe-König prism.

An alternative approach with multi-point sampling is shown in schematicrepresentation in FIG. 4 in which several light sources radiateobliquely into the scanner pupil. Also here a zoom function asrepresented in FIG. 2 can be realized by the utilization of the zoomoptics 41 for imaging between the principal color splitter 17 andscanner 18. By simultaneous beaming of light bundles at different anglesin a plane conjugated to the pupil, light points are generated in aplane conjugated to the object plane which are guided from the scanner18 simultaneously over a partial region of the entire object field.

The image information arises by evaluating all the partial images on aspatially resolving matrix detector 28.

As an additional form of embodiment, a multi-point sampling, asdescribed in U.S. Pat. No. 6,028,306, comes into consideration whosedisclosure is included here relating to this in its full extent. Alsohere a spatially resolving detector 28 is to be provided. The sample isthen illuminated by a multi-point light source which is realized by abeam expander with a micro lens array disposed behind which illuminatesa multi-aperture plate so that a multi-point light source is realizedthereby.

The direction of the line in the sample is then normally predefined, butcan, for example, be changed by rotation.

Here the choice of the inclination of the scanning plane is free, aswell as an arching of the scanning surface in a direction.

Online Representation of the Measured Data on the Display Device (forExample, a Computer Screen)

Through the z-stack according to the invention (in particular in thecase of the line scanner), which can be generated in rapid sequence, anonline representation of forms of representation calculated from atemporal sequence of z-stacks is made possible.

For example, a three-dimensional representation can be offered to usersduring their orientation/navigation in the sample, advantageously from acertain direction of view.

The sample is, in this case, represented in quasi real-time, the timebetween the acquisition of the image and its representation lies on theorder of magnitude of the reaction time of the user, or below it,advantageously at least under one second. [sic] the sample in quasireal-time on the computer screen

For example, a spatial representation of the measured object, inparticular as a shadow projection, can also be done.

By means of different colors (for example, red/blue) or differentdirection of polarization for two represented partial images which theobserver views with colored or polarized glasses, a stereo imagesynthetically generated from two partial images can be presentedimmediately to the user from the very rapidly prepared spatial imagestack.

For example, 3D representation for red-green glasses

For example, 3D representation by means of 3d display aids (3D monitors,for example, with polarized glasses)

For example, 3D projections

For example, 3D surface profile representations and so on

The users can observe events in the sample (for example, caused by theirinteraction) on a three-dimensional image of the sample. The state ofthe art is the online representation of a section through the sample andfollowing the measurement on offline processing of the measured z-stackto form 3D representations.

By changing the function of the focus knob on the stand in az-displacement of the z-range scanned by the z-scanner, the user cannavigate through the sample in all three spatial directions by turningthe z-knob and by the customary moving of the sample tray in x and y andalways obtains directly and online a spatial impression of the measuredsample volume. Additional knobs on the stand (or second allocation, forexample, of the focus knob) could be utilized for the setting of thez-extension of the z-range scanned by the z-scanner. With this, thecustomer can choose the region visible in the z-direction.

An additional knob (or additional allocation of the focus knob) can beutilized for setting the number of z-slices or for setting the distancebetween the scanning planes.

All three aforementioned settings make it possible for the customer thento set said parameters with online observation of the results.

Additional advantages in application are a possible equal temporalresolution in 3 axes, a possible tracking/study/documentation of rapiddynamic processes in 3D.

A rapid 3D-tracking makes possible the tracking of rapid objects orprocesses in the sample in all three spatial coordinates.

Advantageously, an Interactive Three-Dimensional Spline Scan is MadePossible with the Invention

In the preferred variant for an interactive definition of a 3D splinescan a representation of three orthogonal sectional planes of an alreadyacquired image stack of the sample is used. In so doing, the images ofan x-y sectional plane, an x-z-sectional plane, and a y-z-sectionalplane are represented on the computer screen. The position of thesectional planes are marked by lines in the screen display. The user canchange the position of the sectional planes displayed. In so doing, thez-position for the x-y sectional plane, the y-position for thex-z-sectional plane, and the x-position for the y-z-sectional plane canbe chosen freely. The change of position can be done via numericalinput, a slide control, or with a pointer input device.

The preferred variant is a change with a pointer input device. Bymarking a point in one of the three image representation for thesectional planes, the positions for the two other sectional planes aredetermined. With the use of a computer mouse one of the marking linescan be chosen in addition by pressing a mouse key. By moving the mousewith the mouse key pressed the marking line is displaced and thus a newposition of the sectional plane is determined.

The point of intersection of three sectional planes is used for splinedefinition. The user marks several points on the desired spline curve byrepeatedly displacing the sectional planes and activating in each case amarking function via an input device.

In the drawing of FIG. 11 are shown:

-   A1: the display of the x-z section image-   A2: the display of the x-y section image-   A3: the display of the y-z section image-   A4: a defined curve-   A5: the marking of the x-y section plane-   A6: the marking of the x-z section plane-   A7: the marking of the y-z section plane-   A8: the x-y section plane-   A9: the x-z section plane-   A10: the y-z section plane

A simpler variant for spline definition is the use of the display onlyof one sectional plane, preferably the x-y plane, and a capability ofdisplacing in the direction orthogonal to the sectional plane. With apointer device the coordinates in the sectional plane can be determined.

The third coordinate is determined by the position of the displayedimage. Here the definition of points on the spline curve is also done byrepeated displacing and marking the points.

In both variants there is the capability of representing the images ofthe sectional planes also as 3D projection on the monitor. The result ofsuch a projection can appear as in the right part of the image. Todisplace a sectional plane, the user can mark it with the mouse anddisplace it with the mouse button pressed and mouse movement.

Also advantageous is a process with which a qualitatively bettersampling of arbitrarily formed curves (splines) is made possible. Theprocess is particularly suitable for cases where in a curved acquisitionregion (for example, neurons) a high sampling rate is required.

In the acquisition of a curved acquisition region (spline scan) the dataare obtained at present along the sampling points of a predefined curve.This curve is positioned exactly in the sample and corrected by means ofthe position signal. The disadvantage of this process is that processeswhich take place in a small neighborhood of this curve are notregistered. This requires an exact positioning of the curve and staticacquisition conditions free of blurring. In time series and continuousdata acquisition, individual splines in a fluorescence sample arebleached in so doing while the neighboring regions are not bleached.

An advantageous, novel solution will thus be described (see 2):

The data are obtained, as before, along the sampling points of apredefined curve.

This process is repeated many times. In so doing, the sampling pointsare displaced by a small amount in the direction of the normal of thedefined curve with each repetition. The displacement is done in thepositive and negative direction, the amount of the displacement isvariable. Via the data of corresponding sampling points of the curvesthere an average value is formed. In the forming of the average valuethere can be weighting according to the distance of the sampled pointsto the defined curve.

The curves can be defined by spline coordinates by which a spline curveis set (quadratic, cubic spline). With the scanners this curve issampled.

In so doing, the AOTF is controlled so that the sample is illuminated.If the scanner has reached the end of the spline, it is moved withunidirectional image acquisition to the starting point of the displacedcurve following in sequence. During the back-movement the sample is notilluminated by the AOTF control. For a bi-directional image acquisitionthe scanners are moved during the back-movement to the end point of thefollowing spline curve. This shortens the image acquisition time.

A possible variant in the sampling is:

-   -   spline    -   spline displaced +□ in the direction of the normal    -   spline displaced −□ in the direction of the normal    -   spline displaced +2□ in the direction of the normal    -   spline displaced −2□ in the direction of the normal

In this way data are also obtained in a neighborhood of the definedcurve. Through the replacement-side shift of the curve the load on thesample due to bleaching is minimized.

The present laser scanning microscopes have a scanning mode “fast scan”which can be used for finding an object and for resolving. This scanningmode continuously acquires images as rapidly as possible and brings themto the display. For this purpose, the resolution is, in given cases,reduced and the scanning speed set to maximum. With this, a nearlycontinuous image impression results. The disadvantage is that in theconfocal operation only a quite narrow view in the z-direction isdisplayed and thereby objects which lie over or under this plane cannotbe seen.

Also in non-confocal operation the planes over or under the currentfocus plane are imaged only quite unclearly.

In order to represent thick objects universally sharply, there is atpresent the possibility of exposing an image stack (lasts severalseconds) and calculating it offline so that an image results therefromwith increased depth sharpness. That means the individual images of thestack are projected onto one another.

This process is much too slow to use online.

With the help of the fast line scanner described in FIG. 1-4 it isadvantageously possible to acquire up to 200 images per second. Withthis, 5-10 image stacks per second are therefore possible. Aprerequisite is that the z-drive is fast enough, which is the case withthe piezo-objective.

The calculation can also be made fast enough with modern PCs so that atleast 5 images per second can be represented (according to the state ofthe art).

That is fast enough to generate a continuous image impression which issuitable to search for certain objects in the preparation by thepreparation being moved in the xy-direction.

A continuous scan of an image stack is preferably done with a fast linescanner and fast focus operation, an online calculation of theprojection, and representation of the resulting image. The speed can beincreased still further by the xy-images being acquired in bothdirections of movement of the z-scanner (bi-directional z-scan).

The calculation of the resulting image is carried out as follows (FIG.13):

In the individual images of the stack, for all the pixels, the maximumamong the pixels in a stack is determined and this is stored as aresulting pixel in the resulting image.

Pixresult(x,y)-max(pix(x,y,1), pix(x,y,2), . . . , pix(x, y, z)) In thescanning, the representation of the individual images is suppressed andfor this only the result image is displayed.

This new scanning mode can be used for visual inspection of thicksamples as well as input for automatic object recognition programscoming afterwards.

This new scanning mode can, in addition, be implemented for the previousfast scan on LSM with a line scanner.

The determination of regions of interest as described in DE . . . is notadvantageously possible in three-dimensions, for example, for thebleaching of spatial regions.

In the existing confocal or 3D microscopes, regions of interest (ROIs)are 2-dimensional regions in a focus plane of the sample. Moreover, thez-extension of the focus plane in rapidly sampling systems cannot beadjusted since customarily fixed confocal apertures on a disk are used.The object of the invention are applications of a rapidly samplingconfocal or 3D microscope which require simultaneous (real-time) controlof several regions of interest and confocal sectional planes in 3D.

A rapidly sampling confocal or 3D microscope system as described is thusadvantageously equipped with several manipulation and imaging scannersas well as an adjustable confocal aperture and controlled by real-timeelectronics which permit an independent rapid control of the scanningmirror, focus drives, and confocal apertures. With this, newapplications in microscopy are possible which previously, above all inthe study of living samples, were not possible, or possible only to alimited extent:

-   -   1. Analysis of living cells in a 3D-environment whose        neighboring cells react sensitively to laser illumination and        which must be protected from the illumination of the 3D-ROI,    -   2. Analysis of living cells in a 3D-environment with markings        which are intended to be selectively bleached by laser        illumination in 3D, for example, FRET experiments,    -   3. Analysis of living cells in a 3D-environment which are        intended to be selectively bleached by laser illumination and        are also intended to be observed simultaneously outside of the        ROI, for example FRAP and FLIP experiments in 3D,    -   4. Selective analysis of living cells in a 3D environment with        markings and drugs which exhibit the manipulation-related        changes by laser illumination, for example, activation of        transmitters in 3D,    -   5. Selective analysis of living cells in a 3D environment with        markings which exhibit the manipulation-related color changes by        laser illumination, for example, paGFP, Kaede,    -   6. Selective analysis of living cells in a 3D environment with        very weak markings which, for example, require an optimal        balance of confocality against detection sensitivity.

Gimpl, G. et al. have described in 2002 in Prog. in Brain Res. 139:43-55 experiments with ROI bleaches and fluorescence imaging for theanalysis of mobility and distribution of GFP marked oxytocin receptorsin fibroblasts. Therein high demands are placed on the spatialpositioning and resolution as well as the direct temporal sequence ofbleaching and imaging.

Zhang, et al. have described in 2001 in Neuron 31: 261-275 live cellimaging of GFP-transfected nerve cells, where the motion of granuli wasanalyzed by combined bleaching and fluorescence imaging. Therein thedynamics of the nerve cells makes great demands on the speed of theimaging.

Umenishi, F. et al. have described in 2000 in Bipohys. J. 78: 1024-1035an analysis of the spatial motility of aquaporin in GFP-transfectedculture cells. For this, in the cell membrane points were selectivelylocally bleached and the diffusion of the fluorescence in their vicinitywas analyzed.

An optical sectioning through cells at arbitrary tilt angles (2D), forexample, for the optimized representation of branched nerve cells can bedone.

An orthogonal sectioning at very high image rate (X/Z or Y/Z)), forexample, for the study of cytoskeleton dynamics can be done.

A study of fast dynamic process of living cells in 3D, for example, Ca⁺⁻physiology or vesicle transport is made possible.

The study of fast processes at the contact side of cells in monolayercultures, where the optical section runs precisely parallel to thecontact surface, can be done.

FIG. 14 shows a schematic beam path for the arrangement of amanipulation and an imaging scanner on a fast (parallelizing) linescanner.

A linear light source with a y scanner for the movement of the line overthe sample and a point light source with an x/y scanner for thepoint-by-point sampling in the form of a raster of the sample illuminatethe sample via a beam splitter STI.

Both scanners, as well as other elements, are connected to a commoncontrol unit (real-time computer) which realizes a common synchronizedcontrol. That also applies for a fast periodic z-displacement of thefocus device which in common with the X,Y scanners makes possible a fastthree-dimensional irradiation (for example, with the point scanner) andimage acquisition (with the line scanner).

At the beam splitter ST2 the sample light is split in the direction of aline or surface detector in front of which a slotted diaphragm, whichcan be adjusted in a controlled manner relative to its aperture andwhich varies the z-extension of a sample section, is disposed.

FIG. 15 shows a schematic representation of the 2-dimensional scanningregions of the manipulation and imaging scanner on a fast(parallelizing) line scanner.

FIG. 16 shows in schematic representation the 3-dimensional scanningregions of the manipulation and imaging scanner on a fast(parallelizing) line scanner.

Through the line scanner's control represented schematically in FIG. 16,here denoted as scanner 1, a fast image acquisition is carried out instep S depending on the chosen slotted diaphragm aperture which definesthe resolution depth Z and simultaneously a 3-dimensional region ROI 2for image acquisition is sampled, or selectively manipulated, bysuitable wave length manipulations via a second scanning module (scanner2). Imaging and manipulation can, in so doing, also take place indifferent z-planes of the sample by one or more focus optics being setaccordingly. An ROI can be movable and the samples can be guidable foracquisition and/or manipulation of movable samples, where the samplemovement is recorded in three dimensions by means of image processingand corresponding correction signals for the scanner guidance are set.

FIG. 17 shows the representation of a possible form of embodiment of theadaptation of two scanning modules (manipulation and imaging scanners)on one microscope with the use of a lateral and rear port for opticalcoupling. Schematically a central computer/control unit is representedwhich is connected, via signal lines for control and data evaluation, tomodules, coupled to a microscope stand, for image acquisition(preferably line scanner) and for the selective manipulation (scannedmanipulation beam).

The invention described represents a significant expansion of thepossibilities of application of fast confocal laser scanningmicroscopes. The necessity of such a development can easily be seen withthe aid of the standard literature of cellular biology and the cellularand subcellular processes¹ there described and the study methods usedwith a plurality of dyes². See, for example,

-   ¹B. Alberts et al. (2002): Molecular Biology of the Cell; Garland    Science-   ^(1,2)G. Karp (2002): Cell and Molecular Biology: Concepts and    Experiments; Wiley Text Books.-   ^(1,2)R. Yuste et al. (2000): Imaging neurons—a laboratory manual:    Cold Spring Harbor Laboratory Press, New York.-   ²R. P. Haugland (2003): Handbook of fluorescent probes and research    products, 10^(th) Edition; Molecular Probes Inc. and Molecular    Probes Europe BV.

The invention described is suitable, among other things, for the studyof development processes which, above all, are distinguished by dynamicprocesses in the tenth of a second up to 1 hour range. Applicationexamples at the level of united cell structures and entire organisms aredescribed, for example, here: Abdul-Karim, M. A. et al. have describedin 2003 in Microvasc. Res. 66: 113-125 a long-term analysis of bloodvessel changes in living animals, where fluorescence promoters wererecorded at intervals over several days. The 3D-data sets were evaluatedwith adaptive algorithms in order to represent the trajectories ofmotion schematically.

-   -   Soll, D. R. et al. have described in 2003 in Scientific World        Journ. 3: 827-841 a software-based analysis of motion of        microscopic data in nuclei and pseudopodiae of living cells in        all 3 spatial dimensions.    -   Grossmann, R. et al. have described in 2002 in Glia 37: 229-240        a 3D-analysis of the motions of microglial cells in rats, where        the data were gathered over up to 10 hours. Simultaneously, very        fast reactions of the glia also occurred after traumatic injury        so that a high data rate and corresponding data volume arises.

The described invention is excellently suited to the study of internalcellular transport processes since therein quite small motilestructures, e.g. proteins, must be represented at high speed (usually inthe range of hundredths of a second). In order to record the dynamics ofcomplex transport processes, applications such as FRAP with ROI bleachesare also often used. Examples of such studies are, for example,described here;

-   -   Umenishi, F. et al. have described in 2000 in Bipohys. J. 78:        1024-1035 an analysis of the spatial motility of aquaporin in        GFP-transfected culture cells. For this, in the cell membrane        points were selectively locally bleached and the diffusion of        the fluorescence in their vicinity was analyzed.    -   Gimpl, G. et al. have described in 2002 in Prog. in Brain Res.        139: 43-55 experiments with ROI bleaches and fluorescence        imaging for the analysis of mobility and distribution of GFP        marked oxytocin receptors in fibroblasts. Therein high demands        are placed on the spatial positioning and resolution as well as        the direct temporal sequence of bleaching and imaging.    -   Zhang, et al. have described in 2001 in Neuron 31: 261-275 live        cell imaging of GFP-transfected nerve cells, where the motion of        granuli was analyzed by combined bleaching and fluorescence        imaging. Therein the dynamics of the nerve cells makes great        demands on the speed of the imaging.

The described invention is, in particular, suited to the representationof molecular and other subcellular interactions. Therein very smallstructures must be represented with high speed (in the range around onehundredth of a second). In order to resolve the molecule's spatialposition necessary for the interaction, indirect techniques such as, forexample, FRET with ROI bleaches are to be used. Exemplary applicationsare, for example, described here:

-   -   Petersen, M. A. and Dailey, M. E. have described in 2004 in Glia        46: 195-206 a two-channel recording of living hippocampus        cultures of rats, where the two channels are plotted spatially        in 3D and over a rather long time for the markers lectin and        sytox,    -   Yamamoto, N. et al. have described in 2003 in Clin. Exp.        Metastasis 20: 663-638 a two-color imaging of human        firbrosarcoma cells, where green and red fluorescent protein        (GFP and RFP) were observed simultaneously in real-time,    -   Bertera, S. et al. have described in 2003 in Biotechniques 35:        718-722 a multi-coloring of transgenic mice marked with timer        reporter protein which changes its color after synthesis from        green to red. The image acquisition is done as a fast series        3-dimensionally in the tissue in the living animal.

The described invention is outstandingly well-suited to the study ofusually extremely fast signal transmission processes. These usuallyneurophysiological processes place the highest demands on the temporalresolution since the activities mediated by ions play out in the rangeof hundredths to less than thousandths of a second. Exemplaryapplications of studies in muscle or nerve systems are, for example,described here:

-   -   Brum G. et al. have described in 2000 in J. Physiol. 528:        419-433 the localization of fast Ca⁺ activities of the frog        after stimulation with caffeine as a transmitter. The        localization and micrometer-precise resolution succeeded only        through the use of a fast confocal microscope.    -   Schmidt H. et al. have described in 2003 in J. Physiol. 551:        13-32 an analysis of Ca⁺ ions in nerve cell processes of        transgenic mice. The study of fast Ca⁺ transients in mice with        altered Ca⁺-binding proteins could only be carried out with        highly resolving confocal microscopy since even the localization        of the Ca⁺-activity within the nerve cell and its precise        temporal kinetics plays an important role.

1-44. (canceled)
 45. Process for the observation of at least one sampleregion with a light raster microscope having an illumination axis,comprising the steps of: (a) generating illumination light forilluminating a sample, the illumination light having an illuminationaxis, (b) moving the illumination light and the sample relative to eachother along at least one scanning axis substantially perpendicular tothe illumination axis, using a first scanner, (c) moving a secondscanner at an angle to the plane of the relative movement, and (d)acquiring an image by coupling the movements of the first and secondscanners and performing a three-dimensional sampling movement byillumination into an illumination region of the sample.
 46. Processaccording to claim 45, wherein in step (d) the movements of the firstand second scanners are coupled so that at least one of straight andcurved lines and plane and curved surfaces are scanned, and wherein theat least one of straight and curved lines and plane and curved surfacesextend along at least one scanning direction of the first scanner aswell as along the scanning direction of the second scanner.
 47. Processaccording to claim 45, further comprising the step of calibrating atleast the second scanner with the aid of a precalibration to at leastone of the mass of the sample used and the mass of the objective usedand the mass of the sample tray used for the generation of a controlcurve, and wherein step (c) is carried out with the aid of the acquiredcalibration curve.
 48. Process according to claim 45, further comprisingthe step of generating a raster movement with the aid of at least one ofthe frequency response of a control signal and predefinitions from alook-up table.
 49. Process according to claim 45, further comprising thestep of affecting at least one of position and form and temporal changeof the illumination region and the illumination conditions and the imageacquisition conditions using a detection unit via a control unit. 50.Process according to claim 45, wherein the illumination region is oneof: at least one of an arbitrarily formed and oriented line, at leastone of an arbitrarily formed and oriented surface, and at least one ofan arbitrarily formed and oriented volume element.
 51. Process accordingto claim 45, further comprising the step of (e) generating athree-dimensional image with an image creation time/delay time nogreater than the order of magnitude of a user's reaction time andpresenting the three dimensional image to the user, and furthercomprising at least one of the following steps: (f) setting a sectionthickness of the observed image section, concurrently with thegenerating step (e), (g) performing a sample intervention, concurrentlywith the generating step (e), (h) setting of the illuminationconditions, concurrently with the generating step (e), (i) setting theimage acquisition conditions, concurrently with the generating step (e),and (j) setting one of the user's orientation and navigation in thesample, concurrently with the generating step (e).
 52. Process accordingto claim 51, wherein the represented image is generated from severalimage stacks, where, by intensity comparison in each case from samplelocations stacked in the direction of view, the sample location with thehighest intensity is stored and represented.
 53. Process according toclaim 45, wherein the sample is illuminated via scanners independent ofone another, where the different scanners illuminate at least one of thesame and different sample regions and there is at least one of anindependent and common detection of the sample light.
 54. Processaccording to claim 45, wherein the at least one first and secondscanners are provided at least partially in duplicate in a first andsecond system which are directed at least one of simultaneously andsequentially onto the same samples.
 55. Process according to claim 45,wherein in step (a), the illumination light is a first illuminationlight that serves for image observation, and wherein the process furthercomprises the step of generating a second illumination light forilluminating a region of the sample for influencing the sample. 56.Process according to claim 53, wherein in case of a change of theillumination region by the first or second illumination light of theillumination region of the other illumination is tracked by a commoncontrol unit.
 57. Process according to claim 53, further comprising thestep of influencing several sample regions at least one ofsimultaneously and sequentially by the second illumination light,wherein these sample regions are illuminated and detected by image atleast one of simultaneously and sequentially.
 58. Process according toclaim 45, further comprising the step of predefining a variable spatialsample region for at least one of influencing and sample observation,using an input device.
 59. Process according to claim 45, furthercomprising the step of representing three-dimensional sectional planesthrough the sample.
 60. Process according to claim 58, furthercomprising the step of predefining the sectional planes, wherein thepredefining of the sectional planes is done by the user.
 61. Processaccording to claim 45, further comprising the step ofthree-dimensionally marking several points for the definition of athree-dimensional curve.
 62. Process according to claim 45, furthercomprising the step of observing a sample with one of a Nipkowarrangement, a multi-point illumination arrangement, and a resonancescanner.
 63. Process for the study of development processes using theprocess of claim 45, comprising the further step of: (e) studyingdynamic processes in the tenth of a second up to 1 hour range, at thelevel of united cell structures and entire organisms, using the imageacquired in step (d).
 64. Process for the study internal cellulartransport processes using the process of claim 45, comprising thefurther step of: (e) representing small motile structures with highspeed with ROI bleaches, using the image acquired in step (d). 65.Process for representing molecular and other subcellular interactionsusing the process of claim 45, comprising the further step of: (e)representing very small structures with high speed using indirecttechniques for the resolution of submolecular structures, using theimage acquired in step (d).
 66. Process for studying fast signaltransmission processes using the process of claim 45, comprising thefurther step of: (e) studying neurophysiological processes with hightemporal resolution in studies in the muscle or nerve system, using theimage acquired in step (d).
 67. Process according to claim 45, whereinin step (c), the second scanner is moved perpendicular to the plane ofthe relative movement.
 68. Process according to claim 45, furthercomprising the step of determining at least one of a transmissionfunction and a look-up table for the movement of the second scanner. 69.Process according to claim 45, further comprising the step ofcontrolling at least one of the first scanner and the second scannertemporally periodically.
 70. Process according to claim 46, wherein aground frequency of the control curves of the first and second scannersis the same.
 71. Process according to claim 45, wherein the step ofilluminating several sample points is done simultaneously.
 72. Processaccording to claim 45, further comprising the step of illuminating thesample with a linear light distribution.
 73. Process according to claim53, wherein the line is formed from several adjacent points.
 74. Processaccording to claim 45, wherein in step (c), the second scanner comprisesone of an objective with piezo drive and a rapidly adjustable tray andan adaptive mirror.
 75. Process according to claim 53, furthercomprising the step of generating a second periodic raster movement inthe form of a curve.
 76. Process according to claim 48, wherein thecurve is at least one of sinusoidal, linearly increasing, and linearlydecreasing.
 77. Process according to claim 45, wherein in steps (b) and(c), there is a common synchronized control of the first and secondscanners.
 78. Process according to claim 63, wherein the image isgenerated and represented according to a pre-selected direction of view.79. Process according to claim 64, wherein the direction of view isvaried.
 80. Process claim 45, further comprising the step of settingspatially variable illumination regions using an input device. 81.Process according to claim 51, wherein an illumination region,predefined by the input means, follows a structure to be observed incase of change of position.
 82. Process according to claim 51, furthercomprising the step of manipulating a sample in a set illuminationregion.
 83. Process according to claim 60, further comprising the stepof performing at least one of spatially independent manipulation andimaging, using several scanners.
 84. Process according to claim 45,further comprising the step of optical 3D sectioning carried out usingat least one adjustable confocal aperture shield.
 85. Process accordingto claim 53, wherein in case of a detected change within the observedillumination region by the first or second system, the observedillumination region of the other system is changed by a control unitcommon to both systems.
 86. Process according to claim 45, furthercomprising the step of selecting an illumination region with the aid ofan overview image.
 87. Process according to 53, wherein the regionilluminated for image observation is greater than the region illuminatedfor influencing the sample.
 88. Process according to claim 45, whereinin step (d), illumination of the illumination region is done repeatedly,with a displacement of acquisition coordinates being done once inopposite directions.
 89. Process according to claim 62, wherein step (e)comprises analyzing living cells in a three-dimensional tissue groupwith markers, which exhibit manipulation related changes in color bylaser illumination, in combination with living cells in athree-dimensional united tissue structure with very weak markings, whichrequire a restriction in confocality in favor of detection sensitivity,using the confocal scanning microscope.